The long-term objective is to comprehend the relationships between protein conformation and biological function. Under physiological conditions, protein conformational change is driven by macromolecular association, the binding of ligands or effectors, changes in pH or oxidation state, electronically excited states, or catalytic chemistry. These mechanisms are often mediated by chromatically active cofactors or chromophores that signal the state of the protein. Through integrated high resolution structural, spectroscopic and computational studies, this research aims to test the following 3 hypotheses: i) identifiable features of protein structural chemistry act in chromophore generation and incorporation, ii) the protein environment, through both short and long-range interactions, tunes the chromophore for appropriate biological function, iii) the functional properties of the holo-protein depend upon the synergistic partnership between the chromophore and the polypeptide. Research is focused on chromatically active protein systems representing 3 key types of biological transformations that are conserved across the kingdoms of life and important to human health. The interactions of protein with light are represented by 3 photoactive proteins, the signal-transducing photoactive yellow protein (PYP), green fluorescent protein (GFP), an important biological marker, and cryptochrome (CRY), the blue and UVA light photoreceptor that regulates circadian rhythms in plants and animals. Multi-electron transfer reactions are represented by the enzymes sulfite reductase (SiR), which catalyzes the concerted 6-electron reductions of sulfite and nitrite for assimilation of sulfur and nitrogen into the biosphere; and siroheme synthase, which completes the synthesis and metallation of SiR's siroheme cofactor. Reactions with oxygen are represented by the antioxidant superoxide dismutase (SOD) metalloenzymes CuZnSOD and NiSOD, which protect cells from reactive oxygen radicals, and CCS, the Cu-recruiting and inserting chaperone for CuZnSOD. The functioning of CuZnSOD and CCS in vivo promise insight into the role of mutant human CuZnSODs in familial amyotrophic lateral sclerosis. Overall, the proposed research will contribute to identifying mechanisms of protein/chromophore interaction and functionally-important conformational change fundamental to many systems of biological and medical interest.